203 Insulin Gene Therapy of Diabetes in Mice by Glucose Regulated Helper Dependent Adenoviral Vectors Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ����������� �!����� ���[.]
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levels decreased to the normal range within 2 weeks after treatment
and normal levels were maintained for at least 4 months, whereas
untreated diabetic ob/ob mice remained hyperglycemic We examined
the presence of the LPK-MI genome in various tissues by PCR at
one month after treatment with rAAV-LPK-MI We detected the
LPK-MI genome in all tested tissues including the liver, kidney,
spleen, lung, and heart, with the highest amounts in the liver and
spleen When we examined the expression of MI mRNA in various
tissues by reverse transcriptase-PCR, we found that MI mRNA
was expressed mainly in the liver, suggesting that the expression of
MI mRNA is dependent on the liver-specific L-type pyruvate kinase
promoter activity To determine whether rAAV-LPK-MI-treated
ob/ob mice clear glucose from the blood, we performed glucose
tolerance tests at 6 weeks after rAAV-LPK-MI administration We
found that the glucose level peaked at 30 min after glucose injection
and returned to normal within 180 min, whereas the blood glucose
levels of untreated diabetic ob/ob mice were not normalized In
addition, we performed insulin tolerance tests to determine the effect
of rAAV-LPK-MI treatment on insulin resistance Although insulin
resistance in rAAV-LPK-MI-treated ob/ob mice was significantly
alleviated compared to untreated diabetic ob/ob mice, some insulin
resistance was still present as compared to heterozygous ob (+/-)
mice These results suggest that this insulin gene therapy may have
potential therapeutic value for the treatment of obese type 2 diabetes
Peptide 1 for Gene Therapy of Type 2 Diabetes
Donna Armentano,1 Geoffrey Parsons,1 David Souza,1 Dan Yu,1
Samuel Wadsworth,1 Richard Gregory.1
1 Genzyme Corporation, Framingham, MA, United States.
Glucagon-like peptide 1 (GLP-1) is an incretin hormone produced
in the gut in response to nutrient intake and acts in several ways to
reduce post-prandial blood glucose excursions It is a potent
stimulator of glucose-induced insulin secretion, delays gastric
emptying, suppresses glucagon secretion, improves insulin
sensitivity in peripheral tissues and more recently has been shown
insulinoptropic actions of GLP-1 have made it a promising candidate
for the treatment of type 2 diabetes However, the short in vivo
half-life of GLP-1, due to inactivation by dipeptidylpeptidase IV
(DPPIV), has made protein-based treatments challenging requiring
repeat subcutaneous injections or continuous infusion of peptide
A gene therapy approach using vectors to express a DPPIV resistant
analog is one way to circumvent the rapid turnover of GLP-1 and
need for repeat administration We have developed GLP-1 chimeric
expression vectors encoding a DDPIV-resistant 31-amino acid
peptide linked to leader sequences required for secretion of GLP-1
Plasmid or adenoviral expression vectors were administered via tail
vein to diabetic db/db mice in both the C57BL/KsJ (more severe
diabetes) and C57BL/6 (less severe diabetes) genetic backgrounds
Plasma GLP-1, blood glucose, insulin, body weight and %HbA1c
were monitored over time Constitutive secretion of GLP-1 in vivo
resulted in plasma levels of GLP-1 >1nM which were sustained for
periods longer than 1 month Expression of GLP-1 led to lowering
either fasting or random fed hyperglycemia, and consequently,
%HbA1c in these mice These results demonstrate proof-of-concept
for GLP-1 gene therapy of type 2 diabetes using GLP-1 expression
vectors and provide a means for circumventing the need for
continuous infusion of GLP-1 or subcutaneous injection
Liver by Systemic Administration of a Recombinant Adenovirus Expressing a Furin-Cleavable Mouse Preproinsulin Can Remit Diabetes in Diabetic NOD Mice and Evade Autoimmune Attack
Seungjin Shin,1 Jaeseok Han,1 Hee-Sook Jun,1 Ji-Won Yoon.1
1 Julia McFarlane Diabetes Research Centre, The University of Calgary, Calgary, AB, Canada.
Type 1 diabetes results from the destruction of pancreatic beta cells caused by autoimmune-mediated immune responses Restoration of beta cells by islet transplantation or regeneration of pancreatic beta cells from ductal stem cells are attractive methods for the cure of diabetes; however, it is difficult to overcome the hostile beta cell-specific autoimmune responses that might eventually destroy the transplanted or regenerated beta cells Since the liver is
an attractive target for insulin gene therapy, this investigation was initiated to determine whether transduced, insulin-producing hepatocytes can evade autoimmune attack and remit hyperglycemia
in the nonobese diabetic (NOD) mouse, which spontaneously develops autoimmune diabetes We constructed a recombinant adenovirus expressing a furin-cleavable mouse preproinsulin under the cytomegalovirus promoter and the beta-globin/IgG chimeric intron, which will constitutively express insulin (rAd-CMV-mPPI)
In vitro studies showed that an immortalized hepatocyte cell line transduced with rAd-CMV-mPPI produced biologically active insulin
To test this gene construct in vivo, we administered rAd-CMV-mPPI (5 × 109 particles) intravenously through the tail vein of spontaneously diabetic NOD mice (blood glucose > 400 mg/dl) and found that blood glucose was reduced to normal levels within 2 days after administration and maintained at normoglycemic levels for one month, after which time the experiment was terminated We examined the expression of insulin in various tissues, including the liver, kidney, spleen, stomach, heart, pancreas, and lung, and found that insulin was mainly expressed in the liver, as compared to other organs, suggesting that systemic administration of rAd-CMV-mPPI resulted
in delivery mainly to liver tissue To determine whether these insulin-producing hepatocytes are attacked by existing effector T cells in the recipients, we examined the liver at 7, 14, 21, and 30 days after rAd-CMV-mPPI administration We found that the liver was not invaded by effector cells such as T cells and macrophages When we adoptively transferred splenic lymphocytes from the rAd-CMV-mPPI-treated NOD mice into NOD.scid mice, over 90% of the recipients developed diabetes within 7 weeks, indicating that effector cells in the rAd-CMV-mPPI-treated NOD mice were active Based
on these observations, we conclude that constitutive expression of insulin in the liver by systemic administration of rAd-CMV-mPPI can remit diabetes and evade autoimmune attack in diabetic NOD mice
by Glucose-Regulated Helper-Dependent Adenoviral Vectors
Paola Corbella,1 Laura Perani,1 Francesca Mingozzi,1 Alessandra Recchia,1 Fulvio Mavilio,3 Maria-Grazia Roncarolo,1 Luca Falqui.1,2
1 San Raffaele-Telethon Institute for Gene Therapy, San Raffaele Hospital Scientific Institute, Milan, Italy; 2 Medicine,
Endocrinology and Diabetes Unit, San Raffaele Hospital Scientific Institute, Milan, Italy; 3 Department of Biomedical Science, University of Modena School of Medicine, Modena, Italy.
Type 1 diabetes is an autoimmune disease characterized by destruction of insulin-producing pancreatic beta cells, resulting in
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Copyright © The American Society of Gene Therapy
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deficient insulin secretion and hyperglycemia Despite intensive
insulin therapy, properly controlled glucose values are hardly
achieved, therefore degenerative chronic complications may arise
In search for alternative therapeutic options, this project aims at
engineering non-beta cells for the production of insulin in a
glucose-regulated manner The liver is the candidate target organ for this
purpose, as hepatocytes are able to ‘sense’ extracellular glucose
variations and modulate consequentially gene expression To obtain
efficient insulin expression in the liver, we are exploring
Helper-Dependent Adenoviral Vectors (HD-AdV), which should also
provide a better safety profile We generated a vector coding for
Furine cleavable-Human Proinsulin (FurHPI) under the control of
the glucose-responsive liver-specific Pyruvate Kinase gene promoter
(L-PKp) coupled to the SV40 enhancer The vector was injected
into STZ-induced diabetic immunodeficient’nude’ mice (n=4/group)
at two different doses: 1.4x10e11 (low dose) or 2,8x10e11 (high
dose) vp/mouse Human insulin was detectable as early as the first
week after virus injection in both groups, reaching a maximum
group and of 129-192 μU/ml in the high-dose group, and remaining
at similar levels thereafter Mice injected with the low dose remained
hyperglycemic whereas with the high dose blood glucose returned
to normal value, starting from the 2nd week At 4 weeks, the high
dose produced exceedingly high insulin levels (normal insulin values
level was reduced to moderate-severe hypoglycemic value (53±8
mg/dl) Glucose loads and fasting tests demonstrated that L-PKp
displays a slow and weak (3.6-fold over 4 h) induction and 6-fold
repression of insulin synthesis upon glucose increase/decrease We
then generated a second vector containing a Hybrid(L-PK/Spot14)
promoter, which displayed a stronger response to glucose than the
L-PKp once transfected in rat hepatocytes We linked this promoter
to the nuclear LacZ reporter gene in order to quantify liver expression
and tissue specificity This vector was injected into ‘nude’ mice
(n=2) at the dose of 2,8x10e11 particles/mouse Animals were
sacrificed and tissues collected 4 weeks post-gene transfer and
activity was detected in liver extracts only, indicating that this
promoter maintains liver-restricted expression Hepatocytes toxicity
was determined by liver enzyme function test AST and ALT peaked
(x5) at day 1 after injection, then returned to normal level at day 7
The reporter gene will be replaced with FurHPI to obtain faster
glucose-responsive induction of insulin synthesis in liver cells These
results indicate that HD-AdVv are suitable vectors to pursue a
fine-tuned glucose-responsive insulin expression in the liver for gene
therapy of type 1 diabetes
Interleukin-10 (IL-Interleukin-10) for Prevention of Type 1 Diabetes in
NOD Mice: Comparison of Mutant (I87A
Substitution) to Recombinant Murine IL-10
Matthias H Kapturczak,1 Clive H Wasserfall,2 Scott Loiler,3
Cross,2 James M Crawford,2 Tamir M Ellis,2 Terence Flotte,3
1 Medicine, University of Florida, Gainesville, FL; 2 Pathology,
Immunology and Laboratory Medicine, University of Florida,
Gainesville, FL; 3 Pediatrics, University of Florida, Gainesville,
FL.
Type 1 diabetes results from the autoimmne destruction of the
underlying this process are unclear, immunoregulatory defects appear
to be associated with genetic susceptibility and disease progression
Previous studies have demonstrated that a single intramuscular (I.M)
injection of recombinant adeno-associated virus (rAAV) vector (serotype 1) containing the murine IL-10 gene, in a dose and time-dependent fashion, consistently and dependably prevents type 1 diabetes and insulitis development in female non-obese diabetic (NOD) mice However, histological evaluation of the muscle injection sites receiving rAAV-IL-10 revealed long term evidence of myositis and atrophic muscular changes Additional studies have suggested that a substitution of isoleucine at position 87 of IL-10 with alanine inhibits some of the immunostimulatory functions of IL-10, rendering the mutated molecule a potentially more safe agent for studies aimed
at disease prevention To compare IL-10 with I87A-IL10 in terms
of diabetes prevention and side effect profiles, groups of 4 week-old female NOD mice (n=13 animals per group) were I.M injected with
109 infectious units (IU) of rAAV-IL-10, 109 IU of
(encoding the first 33 amino acids of IL-10 and not biologically active) and saline as controls Urine glucose levels were monitored weekly for disease development, with disease rates assessed by life-table (Kaplan-Meier) analysis At 14 weeks post injection, 3 animals from each group were sacrificed for histological and immunological assesments At 38 weeks post injection, diabetes frequency was as follows: IL-10 0% p=0.0016 vs saline, 0.0671 vs delta-IL-10), 109 IU I87A 20% NS vs controls), 107 IU I87A 60%, delta-IL-10 30% and saline 50% In terms of the prevention of insulitis, the degree of infiltration essentially mirrored that of diabetes development with high-dose IL-10 proving most effective.Unlike the saline and delta-IL-10 groups, the IL-10 group and both I87A-IL-10 groups showed development of significant infiltrate of the muscle at the injection site Phenotyic analysis (CD3, CD4, CD8, macrophage, B-lymphocyte) revealed this inflitrate to be predominantly B-lymphocyte in origin Significant muscle fiber atrophy, however, was seen only in the IL-10 group Our study demonstrates that I87A-IL-10, was less efficient than IL-10 in terms
of diabetes prevention Furthermore, such substitution did not abrogate the chemotactic properties of IL-10 Studies of other mutations in the IL-10 structure will be necessary to select for only the desirable parts of the IL-10 function repertoire; allowing for effective disease prevention and devoid of sequence allowing for potentially deleterious inflammation
for the Treatment of Lysosomal Storage Disease
in the Brains of MPS I Mice
Gordon Watson,1 Jacob Bastacky,1 Steve Jungles,2 Michael Vellard,2 Pavel Belichenko,3 Emil Kakkis.2
1 Children’s Hospital Oakland Research Institute, Oakland, CA;
2 BioMarin Pharmaceutical, Novato, CA; 3 Stanford University, Stanford, CA.
Mucopolysaccharidosis Type I (MPS I) is caused by an inherited deficiency of α-l-iduronidase (IDU) The result is a progressive, lysosomal storage disease that includes CNS as well as systemic involvement Enzyme replacement therapy by periodic intravenous infusion of purified IDU has been shown to be effective in treating the non-CNS manifestations of the disease; however, the blood brain barrier excludes therapeutic levels of IDU in the brain Thus, our goal was to develop gene therapy for MPS I specifically targeting the brain Although the blood brain barrier also excludes viral vectors that are administered intravenously, initial studies with a similar storage disease, MPS VII, indicated that intrathecal administration
of an AAV vector could circumvent this problem For MPS VII experiments, which eliminated storage vacuoles throughout the brain,
a relatively high dose of 5 x 1011 vector particles per adult mouse was used To treat MPS I mice, AAV-IDU vectors with and without the woodchuck hepatitis virus posttranslational regulatory element (WPRE) were constructed These vectors contained human IDU